Disease severity and efficacy of homologous vaccination among patients infected with SARS‐CoV‐2 Delta or Omicron VOCs, compared to unvaccinated using main biomarkers

Abstract From March 2021, various countries including Iraq issued prompted recommendations for increased COVID‐19 vaccine protection in individuals especially those at risk of catching the virus (i.e., lifestyle, health sector workers, and chronic diseases). It is critically important to understand the impact of COVID‐19 vaccinations with the most commonly used vaccines (Pfizer and AstraZeneca) among populations either on the severity of the disease or the transmissibility of SARS‐CoV‐2 variants of concern (VOCs) and in sequential waves. This study was conducted to establish the clinical severity of COVID‐19 caused by Delta and Omicron SARS‐CoV‐2 variants among patients who either attended or were admitted to hospitals and to compare the effectiveness of Pfizer and AstraZeneca COVID‐19 vaccines (single or double doses) at least to prevent hospitalizations if not eradicating the pandemic. A case–control study was done of 570 hospitalized patients; including 328 COVID‐19 confirmed patients (166 males, 160 females) who received homologous vaccinations and 242 unvaccinated patients (128 males, 114 females) during the studied waves. The study showed that unvaccinated COVID‐19 patients in both waves had expressed significantly a higher number and longer periods of symptoms than vaccinated ones. Additionally, there was no significant effect of vaccine types, Pfizer and AstraZeneca or vaccine shot numbers on the PCR‐Ct in the last (Omicron) wave of the pandemic. However, in the previous (Delta) wave of the pandemic, fully vaccinated (double doses) COVID‐19 patients had higher PCR‐Ct values. Whether among vaccinated or unvaccinated patients, lower CRP levels recorded during the Omicron wave than that of the Delta wave, and regardless of the vaccine type or shot numbers, there were no significant differences between the two waves. Lower WBCs were observed in patients (vaccinated and unvaccinated) infected with the Delta variant in comparison to those infected with the Omicron variant and without any remarkable effect of the vaccine type or shot numbers. This is the first molecular and investigational study of the Delta variant and circulated Omicron in Iraq, regarding the severity of these two waves of SARS‐CoV‐2 pandemic and the efficacy of homologous vaccination, indicating the insufficiency of two doses and the demand for booster dose(s) as the most effective way of keeping on the safe‐side against SARS‐CoV‐2.


| INTRODUCTION
In the mid-November 2019, the Chinese press issued a warning that Wuhan city was attacked by a new, contagious, and life-threatening viral disease, later named Coronavirus disease 2019  caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The outbreak like wildfire was disseminated over the world in a short span of time, compelling the World Health Organisation (WHO) to declare it a global pandemic in early 2020. 1,2 The disease has continued to exhibit devastating consequences resulting in more than 5.8 million deaths worldwide in Feb. 2022 3 emerging as the most global health crisis since the era of the influenza pandemic of 1918. 4 In South Africa, and in a hospital system, when researchers conducted comparison research for the health outcomes throughout four sequential waves of COVID-19, they defined every wave as the duration of time when positivity proportions outreached 26%. 5 It appears that Omicron is with a much higher average of asymptomatic cases over other variants of concern (VOC), which might interpret its widespread, quick prevalence, even among inhabitants with high previous percentages of SARS-CoV-2 infection. 6 Concerning signs and symptoms of infection with Omicron and the difference to the infection with prior SARS-CoV-2 variants, it has been reported that besides a headache, runny nose, fatigue (both mild and severe), sore throat, sneezing, as the five most known symptoms for Omicron, and the newly announced anorexia and brain haze (more popular in individuals who were completely vaccinated and boosted), extra reports indicated night sweats as well. 7 It is obvious that regardless of the difference between asymptomatic and symptomatic COVID-19, a meaningful part of asymptomatic natural attacks stimulates humoral immune response providing the capability to resist reinfection. 8 Substantial progress in clinical research has led to a better understanding of the management of SARS-CoV-2, limiting the spread of the virus and its variants has become an issue of increasing concern, as SARS-CoV-2 continues to wreak havoc across the world, with many countries enduring a four-wave of outbreaks, mainly due to the emergence of mutant variants of the virus, 9,10 that may have different characteristics than its ancestral strains. Among SARS-CoV-2 variants, only a few are considered VOCs by the WHO, given their impact on global public health as of December 11, 2021, five (alpha, beta, gamma, delta, and Omicron) strains of SARS-CoV-2 have been identified by WHO as VOCs since the beginning of the pandemic. [11][12][13] From mid-2021 to march, 2022, two waves of COVID- 19  After 2 years of the COVID-19 pandemic, health systems worldwide have still not achieved control of the disease. SARS-CoV-2 is highly transmissible with a potential secondary attack rate of more than 17%. 16 This rate of transmission has been reported to be even higher in circulating VOC such as the B.1.1.7 than in preexisting variants. 17 Since its emergence in December 2019, the SARS-CoV-2 virus has infected more than 535 million people and led to at least 6 million deaths globally. 18 In addition to the high disease burden, the virus has brought an unprecedented downpour of social and economic setbacks, the course of which cannot start to be reversed until herd immunity, natural or artificial, is achieved. While six vaccines are already licensed, we are still far from herd immunity, given that vaccines need to be produced at scale, priced affordably, and allocated globally to be widely deployed. 19,20 Expeditious identification of clinical hazard factors, underlying health conditions or comorbidities, and serious consequences which can anticipate advancement in the vicinity of the severe form of the illness (COVID-19) is preeminent for on-time intervention to hinder fatal outcomes. 21,22 Additionally, in consideration of emerging variants and reports of recurrent SARS-CoV-2 infection, the global battle against the virus is far from being over. 23,24 COVID19 vaccination is recognized to be adequate to eradicate the pandemic burden. The population's willingness is to control vaccination programs related to their vaccine acceptance. Limited studies are published highlighting this acceptance. 25 In the present study, we aimed to assess the efficacy of homologous COVID-19 vaccination on the severity of the disease and the transmissibility of SARS-CoV-2 among patients infected with SARS-CoV-2 Delta or Omicron VOCs.  severe type: complicated with any of the following: respiratory distress; respiratory rate ≥ 30 beats/min, mean oxygen saturation ≤ 93% at rest, or ratio of the partial pressure of arterial oxygen to the fraction of inspired oxygen (PaO 2 :FiO 2 ) ≤300 mmHg (1 mmHg = 0.133 kPa).

| Reverse-transcription polymerase chain reaction (RT-PCR)
For detection of SARS-COV2, real-time PCR was carried out using the extracted viral RNA template from the nasopharyngeal swab sample, the procedure was carried out in previous work. 28

| Ethics declarations
All methods were carried out in accordance with relevant guidelines and regulations. We confirm that all experimental protocols were approved by the Ethics Licensing Committee of the Kalar Technical Institute at the Sulaimani Polytechnic University (No. 03 on 02/03/ 2021). In addition, informed consent was obtained from all participants or from a parent or legal guardian if participants were under the age of 18.

| Statistical analysis
A two-way analysis of variance test by GraphPad Prism 9.3 was used to observe the statistical differences between the control and test group for all parameters. In addition, Analyse-it software in Microsoft Excel 2020 was used to perform Principal Component Analysis (PCA)-Biplot to see any correlation between patient comorbidities and treatments with COVID-19 patient groups.
Unvaccinated COVID-19 patients in the 3rd waves had expressed a significantly (pv < 0.0001) higher symptom (2.26 ± 0.53) than vaccinated COVID-19 patients (2.01 ± 0.69); 4th wave, mean ± SD). Interestingly, there were not any significant differences between vaccinated and unvaccinated patients in the 4th wave of the pandemic. Generally, the symptoms in the 4th wave were significantly less than in the 4th wave ( Figure 1D). wave. Interestingly, the symptomatic period in the 4th wave was significantly (pv < 0.0001) shorter than the 3rd wave of pandemics in vaccinated and in unvaccinated patients ( Figure 1E).
In the current study, there were not any significant differences between unvaccinated COVID-19 males and females in both waves for all studied parameters, except for the PCR-Ct value in the 3rd wave where males had a significant (pv = 0.01) higher PCR-Ct value   The current study shows no effect of differences in vaccine types    almost identical mean Ct values to individuals infected without vaccination. 37 Our current findings expand upon prior work, 38 that in infected patients, vaccinated and unvaccinated, the peak viral load appears to be similar, with the risk of potential implications for ahead transmission, due to the strong connection between peak Ct values and infectivity. To make a comparison between omicron and delta infections, a Scottish study determined November 23 to December 19, 2021, as the date was the first case of Omicron reported by the national surveillance data. 39 It is agreed that the overall number of infections is presumed to decrease significantly as the number of individuals acquiring natural immunity increases, which in turn must minimize the rate of transmission. However, an exception of genetic modifications in the circulating variants including 15 and 11 mutations in the receptor-binding domain (RBD) and the N-terminal domain (NTD), respectively, may increase the virus transmissibility, the chances of reinfection, and fractional resistance to the ongoing vaccines, but inherently with less severity. [40][41][42][43][44] Considering biomarkers as explanatory variables, such as SARS-

CoV-2 patients' CRP levels and WBCs were measured in Delta and
Omicron of the current study. It appears that SARS-CoV-2 induces an "imbalance host immune response" 45  years) and 149 individuals aged 12 years and over, mostly vaccinated, recorded with higher peripheral blood lymphocyte counts, and higher normal CRP rate. 40 These data support our findings.
It has been found that vaccine effectiveness after a single dose of either Pfizer or AZ was notably lesser among individuals with the Delta variant (30.7%). However, only the slightest differences in vaccine effectiveness were identified with the Delta variant after two doses of either vaccine. 56 There is a concern that the number of mutations on the S protein of the Omicron variant, may confer the capability of this variant to weaken vaccine effectiveness, and theory suggests that the potency of Abs produced through vaccination will be diminished. 56 Recent scientific knowledge proves that Omicron markedly escapes vaccine-induced immune response after an initial vaccination regimen with Pfizer and AZ vaccines and displays higher infectivity, raising the prospects for increased transmissibility. 31,57 Moreover, in vitro studies using sera COVID-19 patients with a history of hypertension, diabetes, cardiovascular disease, obesity, chronic lung disease, chronic kidney diseases, cancer, and elderly patients in long-term care units are at high risk of contracting the virus and have a worse disease prognosis, even the risk of death among these groups. 21,22,59 Moreover, each antibiotic, antiviral drugs, and glucocorticoids were with a significantly higher incidence of treatment in COVID-19 patients. 20 To eradicate the possibility of the effect of morbidity and treatment on our study groups, a PCA of possible confounding factors including comorbidities and treatments in correlation with studied groups has been performed. Our data has not revealed any significant correlations between underlying comorbidities (single and multiple) or specific treatment with any studied group in comparison with other groups; vaccinated, unvaccinated, Delta, and Omicron.
To conclude, the emergence of Omicron illuminates the challenges facing all types of vaccines, as their designs were based on the genomic sequence of the wild-type strain of the virus from Wuhan. 31 The risk of hospitalization among individuals infected with Omicron vaccinated with double doses was quite similar to among individuals infected with Delta variant, emphasizing the significance of booster doses launch. 60 Moreover, the spike of the Omicron variant increases the tendency for reverse zoonosis, and is with high potential compared to other variants to initiate SARS-CoV-2 animal reservoir, 40 and reduces the chances of SARS-CoV-2 ever being eradicated. Additionally, in consideration of emerging variants of recurrent SARS-CoV-2 infections, the global battle against the virus is far from being over. 23 Future lines of research must focus on the efficacy of full vaccination, booster (third and fourth) doses with heterologous vaccines, and perform genomic sequencing in all SARS-CoV-2 cases. We highly recommend human society, in particular, the health sector, consider the outcomes of this study, and the risk of comorbid conditions associated with the pandemic.

AUTHOR CONTRIBUTIONS
All authors conceived of the idea and planned the experiments; Ayad M. Ali performed the lab work with data collection. Hassan M.
Rostam has performed data analysis. All other authors equally participated in writing, reviewing, and editing the manuscript.

CONFLICT OF INTEREST
The authors declare no conflict of interest.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.